For the past 15 years our translational research program has been developing a multi-modal, gene therapy- based approach for the treatment of cancer. We have evaluated the toxicity and preliminary efficacy of our investigational approach in five clinical trials of non-metastatic prostate cancer. Our early stage results indicate that our approach is safe and has the potential to improve local tumor control. Although local tumor control is important, new therapies for high-risk prostate cancer must also target metastatic disease if they are to have an impact on survival. Hence, we have added a fourth modality to our investigational approach by generating a third-generation adenovirus armed with interleukin 12 (IL-12) that has the potential to eradicate both local and metastatic disease. This new adenovirus has generated encouraging preliminary results in preclinical studies, and we plan to move it into the clinic targeting high-risk prostate cancer. In specific aim 1, we will test the hypothesis that IL-12 will improve the efficacy of replication-competent adenovirus-mediated suicide gene therapy and radiation in an immune-competent, orthotopic model of prostate cancer. C57BL/6 male mice bearing intraprostatic TRAMP-C2 tumors will receive an intratumoral injection of Ad5-yCD/mutTKSR39rep-mIL12 followed by 2 weeks of 5-fluorocytosine (5-FC) + ganciclovir (GCV) prodrug therapy and pelvic radiation. Primary endpoints are local and metastatic tumor control. Secondary endpoints include T cell activation, NK and CTL activity, serum and tumor cytokine levels, and development of anti-tumor immunity. In specific aim 2, we will test the hypothesis that cyclophosphamide (CP) can be combined safely with replication-competent adenovirus-mediated suicide and IL-12 gene therapy and that the combined therapies exhibit synergy in vivo. Efficacy will be examined in the immune-competent, orthotopic TRAMP-C2 tumor model without and with CP. Efficacy endpoints are identical to those in specific aim 1. Toxicity will be examined in C57BL/6 male mice and Syrian hamsters, the latter of which are permissive for human adenovirus replication. In specific aim 3, we will test the hypothesis that replication-competent adenovirus-mediated suicide and IL-12 gene therapy can be combined safely with intensity modulated radiation therapy (IMRT) and androgen suppression therapy (AST) in men with newly-diagnosed, high-risk prostate cancer. Fifteen men (5 cohorts, 3 patients each) with high-risk prostate cancer (Stage e T3 or Gleason e 8 or PSA > 20 ng/mL) will receive a single injection of Ad5- yCD/mutTKSR39rep-hIL12 at five dose levels (1 x 1010 vp to 1 x 1012 vp in half-log increments). The adenovirus will be injected intraprostatically under transrectal ultrasound-guidance. Two days later, men will receive 2 weeks of 5-FC + valganciclovir (vGCV) prodrug therapy concomitant with 80 Gy IMRT and e 2 years of AST. The primary endpoint is toxicity through day 90. Secondary endpoints are: 1) prostate biopsy at 2 years, 2) freedom from biochemical/clinical failure (FFF), 3) disease-specific survival, 4) overall survival, and 5) serum cytokine levels. We believe this research will have high impact because it may lead to better treatments for aggressive forms of prostate cancer.